bioRxiv preprint doi: https://doi.org/10.1101/438176; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant Boris Bogdanow1,2, Katrin Eichelbaum1, Anne Sadewasser2, Xi Wang1,3, Immanuel Husic1, Katharina Paki2 ,Martha Hergeselle1 , Barbara Vetter4, Jingyi Hou1, Wei Chen1,5, Lüder Wiebusch4, Irmtraud M. Meyer1, Thorsten Wolff2 and Matthias Selbach1,6 1 Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin 2 Unit 17 “Influenza and other Respiratory Viruses", Robert Koch Institut, Seestr. 10, 13353 Berlin, Germany 3 Present address: Division of Theoretical Systems Biology, German Cancer Research Center, 69120 Heidelberg, Germany 4 Labor für Pädiatrische Molekularbiologie, Charité Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany 5 Present address: Department of Biology, Southern University of Science and Technology, Xuanyuan Rd. 1088, 518055 Shenzhen, China 6 Charité Universitätsmedizin Berlin, 10117 Berlin, Germany bioRxiv preprint doi: https://doi.org/10.1101/438176; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. SUMMARY A century ago, influenza A virus (IAV) infection caused the 1918 flu pandemic and killed an estimated 20-40 million people. Pandemic IAV outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular details of this species barrier are incompletely understood. We combined metabolic pulse labeling and quantitative shotgun proteomics to globally monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain. While production of host proteins was remarkably similar, we observed striking differences in the kinetics of viral protein synthesis over the course of infection. Most importantly, the matrix protein M1 was inefficiently produced by the bird-adapted strain at later stages. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Experiments with reporter constructs and recombinant influenza viruses revealed that strain-specific M segment splicing is controlled by the 3’ splice site and functionally important for permissive infection. Independent in silico evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, our data identifies M segment RNA splicing as a viral determinant of host range. 2 bioRxiv preprint doi: https://doi.org/10.1101/438176; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. INTRODUCTION Influenza A viruses (IAVs) are negative-sense, single-stranded RNA viruses with a segmented genome. IAV infection causes seasonal epidemics and sporadically pandemic outbreaks in the human population with significant morbidity, mortality and economic burden. IAVs can infect both mammals (e.g. humans, pigs, horses) and birds (e.g. chicken, waterfowl). However, strains that are replicating in birds typically do not infect mammals and vice versa. Pandemics occur when influenza strains of avian origin with novel antigenicity acquire the ability to transmit among humans 1. Understanding the molecular basis of host specificity is therefore of high medical relevance. The species barriers that hinder most avian IAVs from successfully infecting humans are effective at several steps in the viral life cycle. For example, the avian virus receptor hemagglutinin (HA) recognizes oligosaccharides containing terminal sialic acid (SA) that are linked to galactose by α2,3 2. In the human upper respiratory airway epithelium the dominant linkage is of α2,6 type, to which human-adapted hemagglutinin binds. Despite these differences in receptor binding, many avian viruses are internalized by human cells and initiate expression of the viral genome. Such infections typically lead to an abortive, nonproductive outcome in human cell lines 3–6. Our understanding of this intracellular restriction is still incomplete. One well-established factor is the influenza RNA dependent RNA polymerase (RdRp): This enzyme catalyzes replication of the viral genome and transcription of viral mRNAs 7. Polymerases from avian strains are considerably less active in mammalian cells than their counterparts from mammalian-adapted strains 8,9. A wealth of experimental data described adaptive mutations that alter receptor specificity or 3 bioRxiv preprint doi: https://doi.org/10.1101/438176; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. fusion activity of HA (reviewed in 10) and polymerase activity (reviewed in 11). However, relatively little is known about the contribution of other Influenza A virus genes for permissive versus non-permissive infection 12. A crucial aspect for permissive infection is the correct timing of viral gene expression: IAV proteins are produced at the specific phase of infection when they are needed 13. One example is the M gene, which encodes predominantly two polypeptides: The larger protein, M1, is produced from a collinear transcript. The smaller one, M2, is encoded by a differentially spliced transcript 14. M1 is the matrix protein with multiple functions that encapsulates the viral genome and also mediates nuclear export 15,16. M2 is a proton- selective channel that is an integral part of the viral envelope 17,18. The ratio of spliced to unspliced products increases during infection 19, which reflects the changing demands required for optimal viral replication. Systems-level approaches have provided important insights into the molecular details of host-virus interaction 20. For example, RNAi screens identified host factors required for IAV replication 21–23. Also, interaction proteomics experiments identified many cellular binding partners of IAV proteins 24–26. A number of studies also quantified changes in protein abundance 27–32. However, these steady-state measurements cannot reveal the dynamic changes in protein synthesis during different phases of infection. Early studies used radioactive pulse labeling to monitor protein synthesis in IAV infected cells 6,33. However, radioactive pulse labelling cannot provide kinetic profiles for individual proteins. More recently, stable isotope labelling by amino acids in cell culture (SILAC) emerged as 4 bioRxiv preprint doi: https://doi.org/10.1101/438176; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. a powerful means to study the dynamic proteome 34. SILAC-based pulse labelling methods such as pulse SILAC (pSILAC) and dynamic SILAC can quantify protein synthesis and degradation on a proteome-wide scale 35,36. Moreover, metabolic incorporation of bioorthogonal amino acids such as azidohomoalanine (AHA) provides a means to biochemically enrich for newly synthesized proteins 37. In combination with SILAC, AHA labeling can be used to quantify proteome dynamics with high temporal resolution 38–41. Here, we used metabolic pulse labeling and quantitative mass spectrometry to compare proteome dynamics upon infection of human cells with a human-adapted and a bird- adapted IAV strain. We found that host proteins behaved surprisingly similar but observed striking differences in the production of viral proteins, especially for the matrix protein M1. Follow-up experiments with reporter constructs, in silico studies and reverse genetics identified an evolutionarily conserved cis-regulatory element in the M segment as a novel host range determinant. 5 bioRxiv preprint doi: https://doi.org/10.1101/438176; this version posted October 8, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. RESULTS Quantifying the dynamic proteome of permissive and non-permissive IAV infection To assess species specificity of influenza A viruses (IAVs) we used a model system comparing a low-pathogenic avian H3N2 IAV (A/Mallard/439/2004 - Mal) to a seasonal human IAV isolate of the same subtype (A/Panama/2007/1999 - Pan). While the avian virus is not adapted to efficient growth in cultured human cells and causes a non- permissive infection, the seasonal human virus replicates efficiently. We demonstrated previously that the Pan virus produces >1,000 fold more infectious viral progeny than the non-adapted virus, even though both strains efficiently enter human cells and initiate their gene expression program 32. We reasoned that comparing the kinetics of protein synthesis upon infection with both strains might reveal determinants of species specificity. To this end we performed proteome-wide comparative pulse-labeling experiments by combining labeling with azidohomoalanine (AHA) and SILAC (Stable Isotope Labeling of Amino acids in Cell culture) (Figure 1A):